This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention are human Slit polypeptides. The invention also relates to identifying mesenchymal stem cells (MSCs) or other cells comprising such polypeptides or polynucleotides that encode the polypeptides.
Proteins containing epidermal growth factor (EGF)-like sequences have been shown to play an important role in many aspects of eukaryotic cell control, acting as signals for proliferation, growth inhibition, and differentiation. A common feature of these proteins is their involvement in extracellular events and ligand-receptor interactions. In characterizing genomic DNA identified by cross-hybridization to the sequence coding for the tandem EGF repeats of Notch in Drosophila, a related gene sequence from an unlinked locus that also has EGF repeats was discovered. Isolation and characterization of it; showed a corresponce to the slit locus. Further characterization of the related gene sequence established that null mutations to it would result in disruptions of the embryonic central nervous system (CNS) (Rothberg et al. 1988) Thus, it was shown to be involved in neurogenesis.
The Drosophila slit protein contains two types of repeated amino acid sequences: leucine rich repeats (xe2x80x9cLRRxe2x80x9d) and epidermal growth factor-like repeats (xe2x80x9cEGFxe2x80x9d). Its LRRs are arranged in four groups, each composed of four or five LRRs surrounded by conserved amino- and carboxy-flanking regions. The presence of both the LRRs and EGF-like repeats within a single protein make slit unusual in that such combination is not found in any other type of known protein. The absence of any potential transmembrane domains in a sequence having a typical signal sequence and two known extracellular-associated motifs suggests that the slit locus encodes a secreted extracellular protein. The LRR regions of the slit protein and such regions of related proteins participate in extracellular protein-protein interactions. Further, the EGF areas of the slit protein and such regions of related proteins participate in extracellular protein-protein reactions. Moreover, the slit protein is synthesized and secreted by midline glial cells can be come associated with axons. Among other functions, it influences the differentiation of midline cells from the neuroepithelium.
In accordance with one aspect of the present invention, there are provided novel polypeptides and polynucleotides, more particularly, the polypeptides of the present invention are of human origin and are found in human mesenchymal stem cells. MSCs are the formative pluripotential blast cells found inter alia in bone marrow, blood, dermis and periosteum that are capable of differentiating into any of the specific types of mesenchymal or connective tissues (i.e. the tissues of the body that support the specialized elements; particularly adipose, osseous, cartilaginous, elastic, and fibrous connective tissues) depending upon various influences from bioactive factors, such as cytokines. The polypeptide is designated as human Slit. The human Slit polypeptide according to the present invention, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof, is of use in studing the culturing of MSCs and detection of their differentiation and development into multipotent cells.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding such polypeptides, including mRNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
In accordance with another aspect of the present invention there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to sequences of the present invention.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques which comprises culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence of the present invention, under conditions promoting expression of said protein and subsequent recovery of said protein.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides for identifying human MSCs by utilizing the polynucleotides as probes or by expressing the polypeptides encoded thereby, using such polypeptides to produce an antibody specific for one of the polypeptides and then utilizing the antibody to identify the MSCs. Further such polynucleotides, polypeptides and antibodies may be utilized to aid in the identification of MSCs from other species, as well as to investigate/identify MSC functions in humans or other species. In a preferred aspect of the invention, immunocyto-chemistry is utilized with an antibody specific for a polypeptide according to the invention as a means for monitoring the concentration of the polypeptide according to the invention in a culture solution. The MSCs of the culture may thus be subjected to purification procedures to remove differentiated cells and help to maintain the MSCs in culture.
In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides and a method of employing such antibodies to detect diseases related to an overexpression or under expression of a polypeptide compressing a polypeptide with an amino acid sequence according to the present invention. Such antibodies (or active fragments) may be utilized to monitor the growth of MSCs in a culture or to detect the location of tumors in the body.
In accordance with another aspect of the present invention there is provided a method of diagnosing a disease or a susceptibility to a disease related to a mutation in the nucleic acid sequences and the proteins encoded by such nucleic acid sequences.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.